Insulin resistance is one of the hallmarks in the progression to Type 2 diabetes, however the fundamental mechanisms that underlie insulin resistance are poorly understood. Intramuscular triglyceride (IMTG) appears an important predictor of diabetes as it is high, with low insulin sensitivity in the general population. Paradoxically, endurance trained athletes have a high concentration of IMTG, but are very insulin sensitive, which has been called the "athlete's paradox". Studies in my K award are aimed at determining how IMTG synthesis rates and utilization are different between athletes, controls, and individuals with type 2 diabetes (T2D) during rest, exercise, and recovery. I hope to elucidate differences between groups which may help explain the athlete's paradox. Preliminary data from my K award indicates that in addition to increased insulin action, athletes have decreased saturated fatty acid species within diacylglycerol (DAG), and 2-fold higher fractional synthesis rates for IMTG and DAG. These changes may influence insulin sensitivity within skeletal muscle. Studies proposed in this RO3 will be carried out in primary human cell cultures isolated from athletes, type 2 diabetics and sedentary control subjects in my K award. Primary human cell cultures retain the phenotype of the donor, and will allow a mechanistic evaluation of the potential links between intramuscular lipid saturation and synthesis and insulin action. Specific Aim 1: To determine the role of saturated DAG localization on insulin sensitivity. We hypothesize that saturated DAG localized to membranes decreases insulin sensitivity by increasing PKC activation. Thus, changes in the location of saturated DAG, independent of total concentration, may alter skeletal muscle insulin sensitivity. Specific Aim 2: To determine whether acute changes in IMTG and DAG synthesis influence insulin sensitivity. Our hypothesis is that IMTG and DAG turnover, rather than concentration, is a critical link between intramuscular lipids and insulin action. Thus, acute changes in intramuscular lipid synthesis rates may alter skeletal muscle insulin sensitivity. These studies are vital to determine if either saturated DAG and/or synthesis rates of intramuscular lipid alter insulin sensitivity, or are simply correlated in vivo. Results from these studies will further our understanding of the athlete's paradox, and provide key preliminary data for my first RO1 submission. PUBLIC HEALTH RELEVANCE: Type 2 diabetes is considered one of the greatest health crises of our time, making it vital to understand more about this disease. Fat stored in muscle appears to influence diabetes risk, but exactly how this happens is not clear. This study will examine if the type of fat, or how quickly fat is stored in muscle can influence diabetes risk in muscle cells that are isolated from muscle biopsies from human subjects.